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We previously reported that the retroviral vector intermediate AFP-producing cells (PLC/PRF/5). By the (LNAFW0.3TK) expressing the herpes simplex thymidine reporter gene transfection assay, the activity of the variantkinase (HSVtk) gene under the control of the 0.3 kb human type of the promoter was much higher than that of the wild-␣-fetoprotein (AFP) promoter provided the ganciclovir type of the promoter in both HuH-7 and huH-1/cl.2 cells. (GCV)-mediated cytotoxicity in the high AFP-producing Consistent with this, LNAFM0.3TK infection could sensitize (HuH-7) but not in the low AFP-producing (huH-1/cl.2) huH-1/cl.2 cells, as well as HuH-7 and PLC/PRF/5 cells to human hepatoma cells. In the present study, we con-GCV, but did not affect cell growth of nonhepatoma cells structed the retroviral vector (LNAFM0.3TK) in which the (HeLa). In addition, the bystander effect was achieved HSVtk gene expression is regulated by the variant-type of more efficiently by LNAFM0.3TK infection than the 0.3 kb human AFP promoter with a G-to-A substitution LNAFW0.3TK infection in HuH-7 cells. These results sugat nucleotide −119, a point mutation responsible for heredigest that the variant-type of the human AFP promoter tary persistence of human AFP and the vector was applied ensures the therapeutic gene expression in gene therapy to three human hepatoma cell lines, HuH-7, huH-1/cl.2 and particularly for the low AFP-producing hepatoma cells.

In an attempt to develop gene therapy for thyroid carcinomas, the present studies were undertaken to evaluate in vitro and in vivo therapeutic efficacy and toxicity of herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV) treatment, a widely used prodrug/suicide gene therapy, in human thyroid carcinoma cell lines, FRO and WRO cells, using a means of retrovirus-mediated gene transduction. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the HSV-tk gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) shifted from 250 to 0.5 mg/liter in FRO cells, and from 3,000 to 0.09 mg/liter in WRO cells with therapeutic indexes of 500 and 33,000, respectively. Treatment with 30 mg/liter GCV for 4 days led to complete cell death in HSV-tk tumor cells. Nontransduced cells mixed with transduced cells were also effectively killed by GCV (bystander effect). Low concentrations of GCV, which alone showed little cytotoxicity, enhanced radiation-induced cytotoxicity (radiosensitization). In vivo sc FRO-tk tumor models in nude mice also showed dose- and time-dependent tumor regression. The IC50 was less than 2 mg/kg, and treatment with 100 mg/kg GCV for 2 weeks completely eradicated all tumors. The bystander effect and radiosensitization were also obtained in vivo. These results suggest that the HSV-tk/GCV approach to human thyroid carcinoma cells appears to be very efficacious, with a wide therapeutic range, and exerts a bystander effect and radiosensitization both in vitro and in vivo. Thus, HSV-tk/GCV system, alone or in combination with radiotherapy, may be a promising suicide gene therapy for thyroid carcinomas.

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